[0001] This invention relates to novel compounds having therapeutic properties in themselves,
and being capable of potentiating the efficacy of other therapeutically active compounds,
for example cytotoxic compounds used in the treatment of cancer. The novel compounds
have been shown to possess a cell growth inhibiting property, and in addition to this,
also to increase the pharmacological activity of a conventional paclitaxel formulation
and to make it possible to manufacture a new formulation of paclitaxel, exhibiting
improved solubility and therapeutic efficacy.
Background of the invention
[0002] While the term "chemotherapy" originally had a very broad meaning, encompassing the
treatment of various diseases with chemical agents, it has today a more specific meaning.
In modem language, the term "chemotherapy" usually refers to the use of chemical agents
to destroy cancer cells. Among the chemical agents currently used as anticancer drugs,
most function by impairing the ability of the cancer cells to replicate by interfering
with DNA and RNA activities associated with cell division.
[0003] Paclitaxel is a diterpenoid compound {(2R,3S)-3-Benzamido-3-fenyl-2-hydroxy propionic
acid-[(2aR, 4S, 4aS, 6R, 9S, 11S, 12S, 12aR, 12bS)-6,12b-diacetoxy-12-benzoyloxy-2a,
3, 4, 4a, 5, 6, 9, 10, 11, 12, 12a, 12b-dodecahydro-4,11-dihydroxy-4a, 8, 13, 13-tetramethyl-7,
11-methano-5-oxo-1H-cyclodeca[3,4]benz[1,2-b]oxet-9-yl]ester, the active ingredient
in Taxol
®, Bristol-Myers Squibb} originally isolated from the western yew, a cone-bearing evergreen
tree of the genus
Taxus. Paclitaxel is one example of an important chemotherapeutic agent or anticancer drug
currently in use. It has a wide spectrum of activity against solid tumours: primarily
breast cancer, ovarian, colon and non-small cell lung carcinomas. It binds to the
β-subunit of tubulin, resulting in the formation of stable non-functional microtubule
bundles and thus interfering with mitosis. The drug can also induce apoptosis and
has anti-angiogenic properties.
[0004] Paclitaxel is highly protein-bound, has large volumes of distribution, but poor penetration
into the central nervous system. This compound is primarily eliminated from the body
via hepatic metabolism, and its use is therefore generally precluded in severe hepatic
dysfunction.
[0005] In recent years, considerable emphasis has been given to the development of new formulations
of paclitaxel that are suitable for intravenous administration, in order to address
the solubility and toxicity issues associated with this particular drug. Examples
include dispersed systems such as emulsions, liposomes, mixed micelles prepared by
co-precipitation using bile salts and phospholipids (
Alkan-Onyuksel H, et al. Pharm. Res. vol 2. pp. 206-212, 1994), cyclodextrins, and microspheres. Water-soluble prodrugs such as polyethylene glycol-
and polyglutamate-paclitaxel with promising antitumor activity have also been developed.
[0006] The commercially available product, Taxol
® (a paclitaxel concentrate for preparation of solutions for infusion, marketed by
Bristol-Myers Squibb Co., New York, NY, USA), is currently formulated in a vehicle
containing a mixture of polyoxyethylated castor oil (Cremophor
® EL) and ethanol, in the approximate proportions 1:1 (v/v). Cremophor
® EL, which is a commonly used surfactant for lipophilic compounds, has however been
associated with adverse side-effects, such as bronchospasms, hypotension, and other
manifestations of hypersensitivity particularly following rapid administration. Therefor,
long infusion times, high dilution of the ethanol:Cremophor
® EL solution, and pre-medication (e.g. using corticosteroids, antihistamine, and H2-blockers)
are actions resorted to in order to reduce these adverse effects.
[0007] Furthermore, the commercially available formulation is associated with a number of
difficult technical issues such as stability, including the possibility of drug precipitation
upon dilution, filtering requirements and restrictions regarding the use of PVC-containing
vessels and administration sets. It is thus apparent that there is a need for a new
formulation of paclitaxel that is efficacious and less toxic than the commercial product
and which formulation can alleviate the side-effects and set aside the problems currently
associated with preparation and administration of this drug.
[0008] Further, the small difference between the therapeutic and the toxic concentration
severely limits the clinical usefulness of paclitaxel. The therapeutic efficacy could
be improved by delivering the drug with an appropriate microcarrier system, which
is able to change temporal and spatial biodistribution of the drug. This approach
has been suggested for the highly toxic and poorly soluble amphotericin B, which has
been successfully incorporated into disk-like micelles of cholersteryl sulphate (
Lasic D.D. Nature. Vol. 355, 16 Jan., pp.279-280, 1992). In later years, great deal of effort has been given to the development of polymeric
micellar paclitaxel formulations using amphiphilic diblock copolymers (
K. Kataoka et al., JMS-Pure Appl. Chem. A31 (11), pp. 1759-1769, 1994).
[0009] In one study, using a human cancer cell line model, a new formulation containing
biodegradable amphiphilic diblock copolymer, monomethoxy poly(ethylene glycol)-block-poly
(D,L - lactide) (m PEG-PDLLA) and paclitaxel (Genexol
® -PM) and Taxol
® showed comparable
in vitro cytotoxicity at the same concentrations. The polymeric micellar formulation of paclitaxel
produced an increase in a maximum tolerated dose (MTD) as compared with that of Taxol
® when administered i.p.
in vivo. This formulation was said to have advantages over the commercially available injectable
preparation of Taxol
® in terms of low toxicity levels and increased paclitaxel dose (2 to 3-fold higher
levels) (
Kim S. C. et al., J. Controlled Release, v.72, pp. 191-202, 2001).
[0010] The advantages mentioned by the above authors are related to the slow release of
paclitaxel from the micelles, due to a strong hydrophobic association between paclitaxel
and the high molecular weight m PEG-PDLLA. At the same time, according to the authors,
additional studies of a polymeric micellar formulation, comprising paclitaxel in unusually
high doses will be required to fully characterize the nature of toxicities and especially
the more distant consequences this kind of treatment.
[0011] The present inventors have taken a principally different approach. They have made
available novel compounds, comprising the residues of naturally occurring substances
only. These compounds, numbered I through VI, in themselves have low toxicity. A single
dose i.p. toxicity study in rats was carried out in accordance with the OECD principles
of Good Laboratory Practice. It was found that the compounds I - VI, at a dose level
of 100mg / kg body weight did not produce mortality. The minimal lethal dose is thus
above 100 mg / kg body weight for these compounds (I-VI).
[0012] Considering "chemotherapy" in it widest meaning, i.e. the administration of chemical
agents for the prevention, treatment or alleviation of a medical condition, a manifold
of similar problems arise. It is important to optimise efficacy, e.g. the uptake and
target-specificity of the compound, its distribution in the body and its clearance,
simultaneously as minimising the possible side-effects, risks to medical staff etc.
Also the cost of production, ease of preparation, modes of administration, stability
during storage etc must be taken into consideration. In particular, it is desirable
to be able to increase the solubility and bioavailability of poorly soluble pharmaceutical
agents, increasing their efficacy and reducing their side-effects.
[0013] Considering "chemotherapy" in its more specific meaning, i.e. the use of chemical
agents to destroy cancer cells, it remains an urgent task to make available new substances
and formulations, which at least exhibit improved efficacy and less side-effects,
but preferably also improved characteristics concerning solubility, safety, stability
etc.
[0014] In particular, it is desirable to make available a new formulation of paclitaxel,
exhibiting improved stability, improved efficacy and reduced side-effects, compared
to presently available formulations. Further problems and the corresponding innovative
solutions will be evident from the following description and claims.
Prior art
[0015] DE 40 32 187 (Hermes Fabrik pharmazeutischer Präparate Franz Gradinger GmbH & Co., DE) discloses
various N-Retinoyl-L-aminomercapto compounds and their physiologically acceptable
salts. The compounds are suggested for use in the systemic and topical treatment of
diseases of the mucous membranes. A closer study of the structural formulas reveals
that structural elements, central for the compounds (I - VI) according to the present
invention are absent or different in
DE 40 32 187. Notably, the compounds of
DE 40 32 187 contain a sulphur in oxidation state -2 and -1 respectively. The physiological function,
as well as the physical and chemical properties of these sulphur containing compounds
are determined by this oxidation state. There is also no indication that these compounds
would influence the properties of paclitaxel or other water-insoluble or sparingly
soluble substances.
[0016] Kalemkerian et al., Activity of fenretinide plus chemotherapeutic agents in small-cell
lung cancer cell lines, Cancer Chemother Pharmacol, (1999) 43:145-150. This article describes a synthetic retinoid which is both a potent inducer of apoptosis
in cancer cells, and which may have the capability of enhancing the activity of other
cytotoxic agents. All combination studies were performed with a range of concentrations
of each individual agent and both agents together at a fixed ratio corresponding to
the ratio of the IC
50 values of each agent alone as identified in preliminary experiments. The authors
state that their study does not make it possible to say whether the experimental agents
interacted in a mutually exclusive or mutually nonexclusive manner. The issues of
solubilisation and storage of paclitaxel or other water-insoluble or sparingly soluble
pharmaceuticals is not discussed.
[0017] Zhang et al., An investigation of the antitumour activity and biodistribution of polymeric
micellar paclitaxel, Cancer Chemother Pharmacol, (1997) 40:81-86. In this study, the conventional Cremophor Paclitaxel formulation was compared to
a polymeric micellar paclitaxel, administered by i.p. injection. A biodegradable amphiphilic
diblock copolymer, monomethoxy poly (ethylene glycol) block-poly (D,L-lactide) [mPEG-PDLLA]
was used.The micellar formulation showed very promising results. The advantages mentioned
are however related to the slow release of paclitaxel from the micelles, due to strong
hydrophobic association between paclitaxel and the high molecular weight mPEG-PDLLA.
Further, the toxicity and the long-term consequences of this slow release mode of
administration need to be further studied.
Short summary of the invention
[0018] It has been found that therapeutically active compounds can be dissolved in micelles
of a compound which itself displays the desired therapeutic activity or an activity
favourably interacting with or potentiating the desired activity, and which compounds
exhibit low toxicity. The present invention concerns a method for the synthesis of
sodium salts of retinoyl derivatives, belonging to the group.
N-(
all-
trans-Retinoyl)-
L-cysteic acid (I),
N-(13-cis-Retinoyl)-
L-cysteic acid (II),
N-(
all-
trans-Retinoyl)-
L-homocysteic acid (III),
N-(13-cis-Retinoyl)-
L-homocysteic acid (IV),
N-(
all-
trans-Retinoyl)-
L-cysteinesulfinic acid (V), and
N-(
13-cis-Retinoyl)-
L-cysteinesulfinic acid (VI), which exhibit therapeutic effects
per se, and which in combination with known pharmaceuticals exhibit a synergistic effect.
In combination with cytotoxic or cytostatic pharmaceuticals, said novel compounds
introduce improved possibilities to combat cancer. Further, the present invention
discloses a possibility of making water-soluble formulations of water-insoluble or
sparsely soluble pharmaceuticals, such as paclitaxel, with enhanced pharmacological
activity and improved storage and handling properties.
Description of the invention
[0019] The terms "potentiation" and "potentiating" are used to define an action by which
the therapeutic effect of two or more compounds, given simultaneously or substantially
simultaneously, is greater than the effect of said compounds given separately.
[0020] The term "simultaneously" should in this context be interpreted broadly, i.e. encompassing
both situations where two or more compounds are given in admixture, and situations
where the compounds are administered separately, either via the same or different
routes of administration, at the same time or sequentially, provided that the compounds
exert their therapeutic influence in the body at the same or practically the same
time.
[0021] The term "critical micell concentration" or "CMC" is a measure of the concentration
of a solution component, which represents a critical value above which increasing
concentration of said component forces the formation of micelles.
[0022] The present inventors have surprisingly found that
N-(
all-
trans-Retinoyl)-
L-cysteic acid (I),
N-(13-cis-Retinoyl)-
L-cysteic acid (II),
N-(
all-
trans-Retinoyl)-
L-homocysteic acid (III),
N-(
13-cis-Retinoyl)-
L-homocysteic acid (IV),
N-(
all-
trans-Retinoyl)-
L-cysteinesulfinic acid (V), and
N-(
13-cis-Retinoyl)-
L-cysteinesulfinic acid (VI) are capable of increasing the solubility of sparsely soluble
compounds, as well as potentiating their therapeutic efficacy.
[0024] The molecules of these compounds simultaneously exhibit a hydrophilic and a hydrophobic
part in water solutions. In the form of salts, these compounds are capable of forming
micelles in aqueous solutions at concentrations equal to or higher than the critical
micelle concentration (CMC).
[0025] The present invention makes available the use of the above compounds, in form of
their sodium salts, for the manufacture of a medicament for the treatment of cancer.
[0026] Further, the present invention makes available a pharmaceutical composition comprising
an active substance in a therapeutically effective amount, and one of the above compounds
(compounds I - VI), as sodium salt. In particular, the present invention makes available
a pharmaceutical composition wherein the active substance is a cytotoxic compound,
and the potentiating compound is one of the above compounds (compounds I - VI), as
sodium salt. According to one embodiment of the invention, said active substance is
paclitaxel.
[0027] Tests evaluating the cytotoxicity of the inventive compounds in the concentration
40 nM have shown that better results are obtained in the range 0,005 mg/ml to 5,0
mg/ml of the compounds in saline (initial concentrations). A maximum cell growth inhibition
close to 38% was observed at the initial concentration 1 mg/ml (in saline) before
addition to the adenocarcinoma cultures (the MDA-MB-231 cell line).
[0028] Tests evaluating the cytotoxicity of the inventive compounds in the concentration
range 10
-11 M to 10
-6 M in cultures of MDA-MB-231 cells have revealed the following dependence: An increase
of the concentrations of the inventive compounds led to the enhancement of cell growth
inhibition, achieving a value close to 42% at the concentration 10
-6 M.
[0029] The cytotoxicity of the formulation of paclitaxel/compound (I - VI), and compounds
I through VI alone, was compared with paclitaxel and Taxol
® in cultures of MDA-MB-231 cell line. In the case of paclitaxel and Taxol
®, the cell growth inhibition approached 46% at concentrations close to the IC
50 concentration.
[0030] In particular at the same paclitaxel concentration, the formulation of paclitaxel
and compound I or paclitaxel and compound II, both at a molar ratio of the components
of 1:5, exhibited a surprisingly high cell growth inhibition of 70%. The extent of
cell growth inhibition using the commercially available Taxol
® (positive control) was 45%. Already the cytotoxic action of compounds I or II alone,
at a concentration of 40 nM, was close to 40%. The formulations of paclitaxel and
compound I (or compound II) display an increasing cell growth inhibition within the
molar ratio range 1:3-1:5 (paclitaxel : compound I (or compound II)). When further
increasing the ratio of the components to 1:10, the extent of the cell growth inhibition
remains practically unchanged.
[0031] The inventive formulation of
N-(
all-
trans-Retinoyl)-
L-cysteic acid (I),
N-(13-cis-Retinoyl)-L-cysteic acid (II),
N-(
all-
trans-Retinoyl)-
L-homocysteic acid (III),
N-(
13-cis-Retinoyl)-L-homocysteic acid (IV),
N-(
all-
trans-Retinoyl)-L-cysteinesulfinic acid (V),
N-(13-cis-Retinoyl)-L-cysteinesulfinic acid (VI) in form of their sodium salts and paclitaxel
is prepared as follows: Solutions of paclitaxel and any compound (I - VI) in ethanol
(or other aliphatic alcohol) are first prepared in appropriate concentrations. Then
aliquots of these solutions are mixed to form a mixed solution with the desired molar
ratio paclitaxel : compound (I -VI). The obtained solution can be stored for at least
three months at low temperatures, without noticeable change in the properties of compound
(I -VI). Moreover, the formulation retains its cytotoxic effects during prolonged
storage. Before use, the solution is
evaporated in vacuo to yield a waxy solid which is dissolved in saline or other commonly used vehicle
for intravenous infusion to a patient.
[0032] Taxol
® ( Bristol-Myers Squibb Co.) is a formulation containing Paclitaxel (6mg), ethanol
(396mg) and Cremophor
®EL (527mg). The present inventors have shown that the inventive compounds,
N-(
all-
trans-Retinoyl)-
L-cysteic acid (I),
N-(
13-cis-Retinoyl)-
L-cysteic acid (II),
N-(
all-
trans-Retinoyl)-
L-homocysteic acid (III),
N-(
13-cis-Retinoyl)-
L-homocysteic acid (IV),
N-(
all-
trans-Retinoyl)-
L-cysteinesulfinic acid (V), and
N-(
13-cis-Retinoyl)-
L-cysteinesulfinic acid (VI) have excellent solubility in the commercially available
Taxol
® preparation. It is thus possible to easily improve the conventional paclitaxel formulation
using the inventive compounds. An ethanol solution is prepared of one of
N-(
all-
trans-Retinoyl)-
L-cysteic acid (I),
N-(
13-cis-Retinoyl)-
L-cysteic acid (II),
N-(
all-
trans-Retinoyl)-
L-homocysteic acid (III),
N-(
13-cis-Retinoyl)-
L-homocysteic acid (IV),
N-(
all-
trans-Retinoyl)-
L-cysteinesulfinic acid (V), or
N-(
13-cis-Retinoyl)-
L-cysteinesulfinic acid (VI). The obtained solution is evaporated
in vacuo to give waxy solid, whereupon Taxol
® is added, dissolving the waxy solid. The Taxol
® emulsion forms a liquid system with the compounds (I - VI) even at a molar ratio
of paclitaxel to compound (I - VI) of more than 1:20.
[0033] Tests for evaluating the cytotoxicity of an improved paclitaxel formulation (Taxol
® plus compound I - VI) at the molar ratios of paclitaxel : compound I (through compound
VI) from 1:1 to 1:20, were carried out in cultures of human breast adenocarcinoma
(the MDA-MB-231 cell line). The results of these tests are similar to the results
obtained for the formulation paclitaxel and compound I (through VI) in saline. The
extent of cell growth inhibition for this improved paclitaxel formulation (Taxol
® and compound I - VI) at the molar ratio 1:10 was increased by almost 50% (compared
to that of Taxol
® alone).
[0034] It is thus shown that sparsely soluble therapeutic agents can be made more soluble,
and their therapeutic efficacy potentiated, by presenting twelve new therapeutic formulations
comprising the anticancer drug paclitaxel:
- 1) paclitaxel and N-(all-trans-Retinoyl)-L-cysteic acid in saline,
- 2) paclitaxel and N-(13-cis-Retinoyl)-L-cysteic acid in saline,
- 3) paclitaxel and N-(all-trans-Retinoyl)-L-homocysteic acid in saline,
- 4) paclitaxel and N-(13-cis-Retinoyl)-L-homocysteic acid in saline,
- 5) paclitaxel and N-(all-trans-Retinoyl)-L-cysteinesulfinic acid in saline,
- 6) paclitaxel and N-(13-cis-Retinoyl)-L-cysteinesulfinic acid in saline,
- 7) Taxol® and N-(all-trans-Retinoyl)-L-cysteic acid,
- 8) Taxol® and N-(13-cis-Retinoyl)-L-cysteic acid,
- 9) Taxol® and N-(all-trans-Retinoyl)-L-homocysteic acid,
- 10) Taxol® and N-(13-cis-Retinoyl)-L-homocysteic acid,
- 11) Taxol® and N-(all-trans-Retinoyl)-L-cysteinesulfinic acid, and
- 12) Taxol® and N-(13-cis-Retinoyl)-L-cysteinesulfinic acid.
[0035] These formulations showed both good physical and chemical stability, which is believed
to reduce the effects connected with paclitaxel precipitation upon dilution. This
is also believed to solve the issues related to the stringent requirements regarding
facilities and vessels for preparation and storage of conventional paclitaxel preparations.
[0036] Notably, the compounds (I - VI) have low toxicity, but display significant cell growth
inhibition, the effect increasing in the appropriate concentration ranges.
[0037] The results obtained by the present inventors have laid a foundation for the development
of a technique for large-scale synthesis of the compounds (I - VI), in form of the
Na-salts thereof. The synthesis of the compounds (I-VI) of the invention involves
a direct acylation of the amino groups of L-cysteic acid, L-homocysteic, and L-cysteinesulfinic
acid by mixed carbonic-carboxylic acid anhydride in water-organic medium, containing
Na
2CO
3. The solubility of the sodium salts of the compunds (I - VI) in 2-propanol-water
mixtures make it possible to separate insoluble contaminants (inorganic salts and
starting amino acids). The pure compounds (I - VI) are then obtained by precipitation
from their concentrated solutions in 2-propanol-water using a methanol-2-propanol
mixture.
[0038] The above method of synthesis developed by the inventors makes it possible to produce
sodium salts of the compounds (I - VI) in good yields. The method is simple and timesaving.
The final products can be prepared in pure form, without the need of chromatography.
The compounds (I - VI) in the form of sodium salts can be stored in a solution of
2-propanol-water 2:1 (v/v) or ethanol-water 2:1 (v/v) for at least six months at low
temperatures without any noticeable change in their properties. In order to prepare
the formulations of the inventive compounds (I-VI) with paclitaxel or Taxol
®, the sodium salts of these compounds are easily converted into the corresponding
acidic forms, and dissolved in methanol.
[0039] Tests evaluating the cytotoxicity of the compounds I through VI, in the form of sodium
salts in the concentration range 10
-11 M to 10
-6 M, have been performed in cultures of MDA-MB-231 cells, and revealed the following
dependence: an increase of the concentrations of the inventive compounds led to an
enhancement of cell growth inhibition, achieving a value close to 50% for compounds
I and II; a value close to 35% for compounds III and IV; and a value close to 30%
for compounds V and VI.
[0040] Sodium salts of the compounds I through VI were converted into the corresponding
acidic forms of the compounds and dissolved in methanol, in order to prepare the formulations
paclitaxel/compound (I-VI). At the same paclitaxel concentration, the formulation
of paclitaxel and compound I, or paclitaxel and compound II, exhibited a high cell
growth inhibition close to 70% (close to 45% compared to paclitaxel alone as positive
control); the formulation of paclitaxel and compound III or paclitaxel and compound
IV exhibited a cell growth inhibition close to 60% (close to 30% compared to paclitaxel
alone as positive control); the formulation of paclitaxel and compound V or paclitaxel
and compound VI exhibited a cell growth inhibition close to 55% (close to 25% compared
to paclitaxel as positive control). The molar ratio of paclitaxel : compound (I-VI)
was 1:7.
[0041] Sodium salts of the compounds (I-VI) were converted into the corresponding acidic
forms of the compounds and dissolved in Taxol
® to prepare the formulations Taxol
®/compound (I-VI).
[0042] The extent of cell growth inhibition for the formulation of Taxol
®/compound I or Taxol
®/compound II was close to 75% (close to 50% compared to Taxol
® alone as positive control); for the formulation Taxol
®/compound III or Taxol
®/compound IV, the inhibition was close to 65% (close to 35% compared to Taxol
® alone as positive control); for formulation Taxol
®/compound V or Taxol
®/compound VI, close to 60% (close to 30% compared to Taxol
® alone as positive control). The molar ratio of paclitaxel : compound (I-VI) was 1:10.
[0043] The cytotoxic action was similar to that of compound (I-VI)/paclitaxel formulations
in saline (Table 1):
Table 1. Cytotoxic action of formulations prepared according to the invention
Cell growth inhibition % |
Compounds |
OF |
Formulation |
|
W/SAB |
SAB/W |
|
I, II |
close to 71 |
close to 72 |
close to 75 |
III, IV |
close to 62 |
close to 63 |
close to 60 |
V, VI |
close to 56 |
close to 58 |
close to 55 |
[0044] These formulations compare favourably with Taxol
®, but are believed to remove or alleviate the adverse effects associated to Cremophor
® EL. Tests
in vitro show remarkable results, and there are substantial grounds to believe that the pharmacological
activity in human patients is improved, compared to that of conventional paclitaxel
formulations.
[0045] Consequently, the present inventors disclose a method for preparing a water-soluble
formulation of paclitaxel, comprising the steps of dissolving paclitaxel in a first
solvent, dissolving a compound (I-VI) in a second solvent, mixing the aliquots of
the resulting solutions of paclitaxel and the said compound in a desired molar ratio,
and evaporating the resulting mixture to dryness.
[0046] Further, the inventors disclose a method for preparation a water-soluble improved
formulation of Taxol
®, comprising the step of dissolving a compound (I-VI) in a solvent, evaporating the
desired aliquot of the resulting solution to dryness and dissolving the residue in
Taxol
®.
[0047] Further, the inventors disclose a method for preparing the formulation of paclitaxel
for administration to a patient, comprising the steps of dissolving paclitaxel in
a first solvent, dissolving a compound (I-VI) in a second solvent, mixing the aliquots
of the resulting solutions of paclitaxel and the said compound in a desired molar
ratio, evaporating the resulting mixture to dryness forming a residue of paclitaxel
and the said compound, dissolving the said residue in a aqueous solution, lyophilisation
of the solution formed, followed by reconstitution of the lyophilised product using
a vehicle suitable for administration to a patient.
[0048] The invention will be illustrated in closer detail in the following non-limiting
examples.
Examples
MATERIALS AND METHODS
[0049] all-
trans-Retinoic, 13-
cis-Retinoic, L-cysteic, L-homocysteic, and L-cysteinesulfinic acids were purchased from
Sigma Chemical Co, St. Louis, MO, USA. All other chemical reagents and solvents were
purchased from Aldrich Chemical Co.
[0050] 1H-NMR spectra were determined at 400 MHz using a Varian Unity-400 spectrometer. Spectra
were determinded for the acidic forms of the compounds, however the derivatives of
L-homocysteic acid III and IV were used in the form of sodium salts. DMSO-d
6 was used as a solvent.
[0051] Merck silica gel 60 precoated plates were used for thin-layer chromatography (TLC)
and developed in solvent system of chloroform : methanol : acetic acid : water (65:25:5:5,
v/v/v/v). Detection of the compounds on TLC plates was achieved by spraying with 10%
H
2SO
4 in methanol, heating to 150°C, or using a solution of 0,3 % ninhydrin in 1-butanol-acetic
acid 100:3 (v/v).
[0052] The determination of the concentrations of the synthesized compounds was performed
by UV-spectra (Shimadzu UV-mini-1240 spectrophotometer, λ÷250-500 nm, λ
max 346 nm, ε 45000, MeOH) for the derivatives of
all-
trans-retinoic acid, and by weight for the derivatives of 13-
cis-retinoic acid. The concentrations determined by UV-spectra were equal to the weights
of the samples.
[0053] The compounds synthesized (in acidic form) are soluble in chloroform, THF, ethanol,
methanol, and 70% aq ethanol. Sodium salts of the derivatives of L-cysteic and L-cysteinesulfinic
acids (I, II, V, VI) are soluble only in mixtures containing water (e.g. methanol-water,
ethanol-water, THF-water, etc). Sodium salts of the derivatives of L-homocysteic acid
(III, IV) are also dissolved in methanol and some methanol containing mixtures (e.g.
chloroform-methanol, THF-methanol, etc).
[0054] All steps of the synthesis were performed in dry nitrogen atmosphere.
[0055] Paclitaxel was purchased from Sigma (St. Louis, MO, USA) and Taxol® was purchased
from Bristol-Myers Squibb Co. Human breast adenocarcinoma MDA-MB-231 cell line was
purchased from American Type Culture Collection (ATCC-HTB-26, Lot 1227150). The cell
line was propagated by cultivation in Nunclon 25cm
2 flasks (Nunc A/S, Denmark) in Minimum Essential Medium Eagle (MEM), containing antibiotics
and supplemented with 10% (v/v) fetal bovine serum (FBS) (Sigma, St.Louis, MO, USA).
The cultures were maintained in growth medium at 37°C in a humidified atmosphere,
95% air and 5% CO
2.
[0056] A suspension of tumour cells (60 × 10
3 cells/mL) was prepared in MEM with 5% FBS and antibiotics. Cell suspension (200 µL)
was seeded in wells of Nunclon 96-microwell plates (Nunc A/S, Denmark) at a density
of 12 × 10
3 cell/well. The solutions of the drugs to be tested were added to the cultures on
day 0 or day 1 in volumes of 2 µL. In all cases the cells were incubated for 3 days.
[0057] At the end of the incubation period, adherent cells were detached by trypsinization
and the number of viable cells was counted using trypan blue dye exclusion test and
a hemocytometer.
[0058] All experiments were performed at least twice and the cells counts were done in triplicate
for each drug concentration. Each control and test series consisted of 6-8 cultures.
The results are expressed as mean cell number ± SD and the differences between control
and test series were evaluated by means of Student's t-test. The cytotoxicity of each
tested drug was evaluated by the extent of cell growth inhibition. The Cell growth
inhibition was evaluated as follows:
Example 1. Synthesis of N-(all-trans-retinoyl)-L-cysteic acid (sodium salt) (I)
[0059] all-trans-Retinoic acid (150 mg, 0.5mmol) and triethylamine (71 µl, 0.51 mmol) were dissolved
in 1 ml anhydrous tetrahydrofuran, then anhydrous acetonitrile (2ml) was added, the
mixture chilled to -20°C, and 66 µl (0.51 mmol) of isobutyl chloroformate added. After
30 min, the mixture, free of the precipitated triethylamine hydrochloride, was pipetted
in a solution of L-cysteic acid monohydrate (94 mg, 0.5 mmol) in 3 ml of 1M Na
2CO
3 and 1.5 ml of H
2O. The mixture obtained was stirred for about 5h at 20-25°C, diluted with 15 ml of
2-propanol-water 2:1 (v/v), filtered and concentrated under reduced pressure to approximately
5 ml. 20 ml of 2-propanol-water 5:1 (v/v) was then added, and the solution shaken
during a few minutes while a white precipitate was formed. The suspension obtained
was filtered, concentrated as described above, and diluted with 20 ml of 2-propanol-methanol
1:1 (v/v). A yellow precipitate of the desired product was separated, dissolved in
15 ml of 2-propanol-water 2:1 (v/v) and stored overnight at -18°C in order to remove
traces of impurities. After removal of insoluble material, a clear solution of pure
product was obtained. This solution was used for storage of compound I.
[0061] R
f 0.30-0.35.
1H-NMR (CD
3SOCD
3, 400 MHz) δ 1.00 [6H, s, C(C
H3)
2], 1.43 and 1.56 [2H+2H, 2m, C
H2C
H2C(CH
3)
2], 1.67 [3H, s, C
H3C=CC(CH
3)
2], 1.95 [3H, s, C
H3C=(CH)
3C(CH
3)=CHCO], 1.99 (2H, m, C
H2C=), 2.25 (3H, s, C
H3C=CHCO), 2.78-2.89 (2H, m,
SCH2), 4.40 (1H, m, NC
H), 5.84 (1H, s, =C
HCO), 6.11-6.36 [4H, m, C
H=C
HC(CH
3)=C
HCH=C
H], 6.91 [1H, dd,
J 15.2, 11.5 Hz, CH=CHC(CH
3)=CHC
H=CH], 8.07 (1H, d,
J 6.4 Hz, N
H), ~ 12.4 (1H, br s, CO
2H).
[0062] In order to obtain the acidic form of compound I, a solution of the compound (2-propanol-water
2:1, v/v) was evaporated under reduced pressure, dissolved in water, carefully acidified
with 0.1M HCl to pH 3.5 and extracted with chloroform-2-propanol-methanol (2:1:1,
v/v/v). After evaporation of organic solvents under reduced pressure, the residue
was dissolved in dry methanol (approximately 5 mg/mL), stored overnight at -18°C,
filtered and immediately used.
Example 2. Synthesis of N-(13-cis-retinoyl)-L-cysteic acid (sodium salt) (II)
[0063] This compound was synthesized as described above for I, using 150 mg (0.5 mmol) of
13-
cis-retinoic acid and 94 mg (0.5 mmol) of L-cysteic acid monohydrate.
[0065] R
f 0.30-0.35.
1H-NMR (CD
3SOCD
3, 400 MHz) δ 1.00 [6H, s, C(C
H3)
2], 1.43 and 1.57 [2H+2H, 2m, C
H2C
H2C(CH
3)
2], 1.67 [3H, s, C
H3C=CC(CH
3)
2], 1.95 and 1.98 (3H+3H, 2s, C
H3C=(CH)
3C(C
H3)=CHCO], 1.99 (2H, m, C
H2C=), 2.82 (2H, m, SC
H2), 4.37 (1H, m, NC
H), 5.68 (1H, s, =C
HCO), 6.13-6.26 [3H, m, C
H=C
HC(CH
3)=C
HCH=CH], 6.87 [1H, dd,
J 15.4, 11.4 Hz, CH=CHC(CH
3)=CHC
H=CH], 7.85 [1H, d,
J 15.4 Hz, CH=CHC(CH
3)=CHCH=C
H], 8.04 (1H, d,
J 6.4 Hz, N
H), ~ 12.4 (1H, br s, CO
2H).
Example 3. Synthesis of N-(all-trans-retinoyl)-L-homocysteic acid (sodium salt (III)
[0066] This compound was synthesized as described above for I, using 150 mg (0.5 mmol) of
all-trans-retinoic acid and 92 mg (0.5 mmol) of L-homocysteic acid.
[0068] R
f 0.30-0.35.
1H-NMR (CD
3SOCD
3, 400 MHz) δ 1.00 [6H, s, C(C
H3)
2], 1.43 and 1.56 [2H+2H, 2m, C
H2C
H2C(CH
3)
2], 1.67 [3H, s, C
H3C=CC(CH
3)
2], 1.87 (2H, m, SCH
2C
H2), 1.94 [3H, s, C
H3C=(CH)
3C(CH
3)=CHCO], 1.99 (2H, m, C
H2C=), 2.25 (3H, s, C
H3C=CHCO), 2.41 (2H, m, SC
H2), 3.94 (1H, m, NC
H), 5.98 (1H, s, =C
HCO), 6.11-6.32 [4H, m, C
H=C
HC(CH
3)=C
HCH=C
H], 6.86 (1H, dd,
J 15.0, 11.5 Hz, =CHC
H=CH), 7.43 (1H, d,
J 7.1 Hz, N
H).
Example 4. Synthesis of N-(13-cis-retinoyl)-L-homocysteic acid (sodium salt (IV)
[0069] This compound was synthesized as described above for I, using 150 mg (0.5 mmol) of
13-
cis-retinoic acid and 92 mg (0.5 mmol) of L-homocysteic acid.
[0071] R
f 0.30-0.35.
1H-NMR (CD
3SOCD
3, 400 MHz) δ 1.00 [6H, s, C(C
H3)
2], 1.42 and 1.56 [2H+2H, 2m, C
H2C
H2C(CH
3)
2], 1.68 [3H, s, C
H C=CC(CH
3)
2], 1.87 (2H, m, SCH
2C
H2), 1.93 and 1.94 [3H+3H, 2s, C
H3C=(CH)
3C(C
H3)=CHCO], 1.99 (2H, m, C
H2C=), 2.41 (2H, m, SC
H2), 3.95 (1H, m, NC
H), 5.83 (1H, s, =C
HCO), 6.18 [3H, m, C
H=C
HC(CH
3)=C
HCH=CH], 6.81 [1H, dd,
J 15.4, 11.4 Hz, CH=CHC(CH
3)=CHC
H=CH], 7.42 (1H, d,
J 7.3 Hz, N
H), 7.93 [1H, d,
J 15.4 Hz, CH=CHC(CH
3)=CHCH=C
H].
Example 5. Synthesis of N-(all-trans-retinoyl)-L-cysteinesulfinic acid (sodium salt) (V)
[0072] This compound was synthesized as described above for I, using 150 mg (0.5 mmol) of
all-trans-retinoic acid and 77 mg (0.5 mmol) of L-cysteinesulfinic acid.
[0074] R
f 0.30-0.35.
1H-NMR (CD
3SOCD
3, 400 MHz) δ 1.00 [6H, s, C(C
H3)
2], 1.43 and 1.56 [2H+2H, 2m, C
H2C
H2C(CH
3)
2], 1.67 [3H, s, C
H3C=CC(CH
3)
2], 1.96 [3H, s, C
H3C=(CH)
3C(CH
3)=CHCO], 2.00 (2H, m, C
H2C=), 2.26 (3H, s, C
H3C=CHCO), 2.88-3.00 (2H, m, SC
H2), 4.51 (1H, m, NC
H), 5.84 (1H, s, =C
HCO), 6.11-6.36 [4H, m, C
H=C
HC(CH
3)=C
HCH=C
H], 6.94 [1H, dd,
J 15.0, 11.4 Hz, CH=CHC(CH
3)=CHC
H=CH], 8.47 (1H, d,
J 7.9 Hz, N
H), ~ 12.6 (1H, br s, CO
2H).
Example 6. Synthesis of N-(13-cis-retinoyl)-L-cysteinesulfinic acid (sodium salt (VI)
[0075] This compound was synthesized as described above for I, using 150 mg (0.5 mmol) of
13-
cis-retinoic acid and 77 mg (0.5 mmol) of L-cysteinesulfinic acid.
[0077] R
f 0.30-0.35.
1H-NMR (CD
3SOCD
3, 400 MHz) δ 1.00 [6H, s, C(C
H3)
2], 1.42 and 1.56 [2H+2H, 2m, C
H2C
H2C(CH
3)
2], 1.67 [3H, s, C
H3C=CC(CH
3)
2], 1.95 and 1.97 [3H+3H, 2s, C
H3C=(CH)
3C(C
H3)=CHCO], 1.99 (2H, m, C
H2C=), 2.51 (1H, dd,
J 13.2, 2.9 Hz, SC
HaH
b), 2.65 (1H, dd,
J 13.2, 8.2 Hz, SCH
aHb), 4.77 (1H, m, NC
H), 5.72 (1H, s, =C
HCO), 6.20 [3H, m, C
H=C
HC(CH
3)=C
HCH=CH], 6.87 [1H, dd,
J 15.6, 11.5 Hz, CH=CHC(CH
3)=CHC
H=CH], 7.88 [1H, d,
J 15.6 Hz, CH=CHC(CH
3)=CHCH=C
H], 8.21 (1H, d,
J 7.9 Hz, N
H)
.
Example 7. Evaluation of cytotoxicity of compound I in cultures of human breast adenocarcinoma
MDA-MB-231 cell line, related to final concentration of compound I in cultures
[0078] The sodium salt of compound I was dissolved in saline (1 mg/ml). From this solution
the working solutions of compound I in MEM with 5% FBS were prepared in different
concentrations for adding to cultures, by means of consecutive dilutions.
[0079] Cultures of MDA-MB-231 cells were treated with drug solutions in MEM, containing
5% FBS, after sowing on day 1. Aliquots of working solutions (2 µL) with different
concentrations of compound I were added to 200 µL cultures to a final concentration
of compound I from 10
-11 to 10
-6 mol/L in the cultures. In control cultures, 2 µL of medium with 5% FBS was added
as solvent control. After cultivation for two consecutive days, the number of living
cells in the cultures was counted and the extent of growth inhibition of MDA-MB-231
cells calculated for evaluating the cytotoxicity of the tested solutions of compound
I.
[0080] After three days of cultivation the control cultures contained (58.7 ± 2.24) × 10
3 cells.
[0081] Cultures treated with solutions of compound I had the following number of living
cells:
10-11 mol/L: (41.9 ± 2.17) × 103, cell growth inhibition 28.6% (p < 0.001);
10-10 mol/L: (37.3 ± 2.84) × 103, cell growth inhibition 36.5% (p < 0.001);
10-9 mol/L: (35.4 ± 2.23) × 103, cell growth inhibition 39.7% (p < 0.001);
10-8 mol/L: (31.6 ± 1.69) × 103, cell growth inhibition 46.2% (p < 0.001);
4 × 10-8 mol/L: (31.2 ± 1.72) × 103, cell growth inhibition 46.8% (p < 0.001);
10-7 mol/L: (30.5 ± 0.89) × 103, cell growth inhibition 48.0% (p < 0.001);
10-6 mol/L: (29.5 ± 1.36) × 103, cell growth inhibition 49.7% (p < 0.001).
[0082] It is thus shown, that compound I exerts a significant cytotoxic action against human
breast adenocarcinoma cells. The extent of cell growth inhibition increased to 49.7%
(p < 0.001) by increasing the concentration of compound I.
Example 8. Evaluation of the cytotoxicity of the formulation paclitaxel/compound I
in cultures of human breast adenocarcinoma MDA-MB-231 cell line, related to the molar
ratio paclitaxel :compound I
[0083] The sodium salt of compound I was converted into the acidic form of compound I and
dissolved in methanol. A solution of paclitaxel in methanol and a solution of compound
I in methanol was mixed together. After stirring the organic solvent was evaporated.
The resulting dried film was dissolved in saline.
[0084] Initial solutions of the formulation in saline at the molar ratios paclitaxel : compound
I equal to 1:3, 1:4, 1:5, 1:6, 1:7 and 1:10 were prepared. From these solutions the
working solutions in MEM with 5% FBS were prepared for adding to cultures. The concentration
of paclitaxel was equal to 10
-6 M.
[0085] Cultures of MDA-MB-231 cells were treated with drug solutions in MEM, containing
5% FBS, after sowing on day 1. Aliquots of the working solutions (2 µL) were added
to 200 µL cultures to a final concentration of paclitaxel in the cultures equal to
10
-8 M. In control cultures 2 µL of medium with 5% FBS was added as solvent control. After
cultivation for two consecutive days, the number of living cells in cultures was calculated
for evaluating the cytotoxicity of the tested solutions of the formulation.
[0086] After three days of cultivation the control cultures contained (54.4 ± 2.51) × 10
3 cells.
[0087] Cultures, treated with paclitaxel in concentration of 10 nM, contained
[0088] (29.3 ± 1.13) × 10
3 cells, cell growth inhibition was 46.1% (p < 0.001).
[0089] Cultures, treated with solutions of the formulation at a molar ratio of paclitaxel
: compound I equal to 1:3, 1:4, 1:5, 1:6, 1:7 and 1:10 in medium with 5% FBS, had
the following number of living cells:
1:3: (20.1 ± 1.53) × 103, the cell growth inhibition being 63.1% (p < 0.001), and the cell growth inhibition
compared to that of paclitaxel was increased by 31.4% (p <0.002);
1:4: (18.9 ± 0.92) × 103, the cell growth inhibition being 65.3% (p < 0.001), and the cell growth inhibition
compared to that of paclitaxel was increased by 35.5% (p <0.001);
1:5: (17.4 ± 1.18) × 103, the cell growth inhibition being 68.0% (p < 0.001), and the cell growth inhibition
compared to that of paclitaxel was increased by 40.6% (p <0.001);
1:6: (16.9 ± 1.08) × 103, the cell growth inhibition being 68.9% (p < 0.001), and the cell growth inhibition
compared to that of paclitaxel was increased by 42.3% (p <0.001);
1:7: (15.8 ± 1.34) × 103, the cell growth inhibition being 71.0% (p< 0.001), and the cell growth inhibition
compared to that of paclitaxel was increased by 46.1% (p <0.001);
1:10: (15.2 ± 0.72) × 103, the cell growth inhibition being 72.1% (p < 0.001), and the cell growth inhibition
compared to that of paclitaxel was increased by 48.1% (p <0.001).
Example 9. Comparative cytotoxity testing of Taxol® and an inventive formulation Taxol® + compound I) in cultures of human breast adenocarcinoma MDA-MB-231 cell line related
to molar ratios paclitaxel : compound I
[0090] The sodium salt of compound I was converted into the acidic form of compound I and
dissolved in methanol. The organic solvent was evaporated. The resulting dried film
was dissolved in Taxol
®.
[0091] Initial solutions of the inventive formulation at the molar ratios of paclitaxel
to compound I equal to 1:1, 1:3, 1:6, 1:10, 1:15 and 1:20 were prepared. From these
solutions the working solutions in MEM with 5% FBS were prepared for adding to the
cultures. The concentration of paclitaxel was equal to 10
-6 M.
[0092] Cultures of MDA-MB-231 cells were treated with drug solutions in MEM, containing
5% FBS, after sowing on day 1. Aliquots of the working solutions (2 µL) were added
to 200 µL cultures to a final concentration of paclitaxel in the cultures equal to
10
-8 M. In control cultures 2 µL of medium with 5% FBS was added as solvent control. After
cultivation for two consecutive days the number of living cells in cultures was calculated
for evaluating the cytotoxicity of tested solutions.
[0093] After three days of cultivation, the control cultures contained (54.0 ± 1.60) × 10
3 cells.
[0094] Cultures, treated with Taxol
® in concentration of 10 nM paclitaxel, contained
[0095] (29.0 ± 0.91) × 10
3 cells, the cell growth inhibition being 46.3% (p < 0.001).
[0096] Cultures, treated with solutions of the inventive formulation (Taxol
® + compound I) at the molar ratio paclitaxel : compound I equal to 1:1, 1:3, 1:6,
1:10, 1:15 and 1:20 in medium with 5% FBS, had the following number of living cells:
1:1: (21.5 ± 2.18) × 103, the cell growth inhibition being 60.2% (p< 0.001), and the cell growth inhibition
compared to that of Taxol® was increased by 25.9% (p < 0.01);
1:3: (18.5 ± 1.08) × 103, the cell growth inhibition being 65.7% (p< 0.001), and the cell growth inhibition
compared to that of Taxol® was increased by 36.2% (p <0.001);
1:6: (14.2 ± 0.75) × 103, the cell growth inhibition being 73.7% (p < 0.001), and the cell growth inhibition
compared to that of Taxol® was increased by 51.0% (p <0.001);
1:10: (13.8 ± 0.63) × 103, the cell growth inhibition being 74.4% (p < 0.001), and the cell growth inhibition
compared to that of Taxol® was increased by 52.4% (p <0.001);
1:15: (13.4 ± 1.22) × 103, the cell growth inhibition being 75.2% (p < 0.001), and the cell growth inhibition
compared to that of Taxol® was increased by 53.8% (p <0.001);
1:20: (13.5 ± 1.14) × 103, cell growth inhibition being 75.0% (p < 0.001), and the cell growth inhibition compared
to that of Taxol® was increased by 53.4% (p <0.001).
Example 10. Evalution of the cytotoxicity of compound II in cultures of human breast
adenocarcinoma MDA-MB-231 cell line related to the final concentration of compound
II in cultures
[0097] The sodium salt of compound II was dissolved in saline (1 mg/ml). From this solution,
the working solutions of compound II in MEM with 5% FBS in different concentrations
were prepared by means of consecutive dilutions for adding to the cultures.
[0098] Cultures of MDA-MB-231 cells were treated with drug solutions in MEM, containing
5% FBS, after sowing on day 1. Aliquots of working solutions (2 µL) with different
concentrations of compound II were added to 200 µL cultures to a final concentration
of compound II from 10
-11 to 10
-6 mol/L in the cultures. In the control cultures, 2 µL of medium with 5% FBS was added
as solvent control. After cultivation for two consecutive days, the number of living
cells in the cultures was counted and the extent of growth inhibition of MDA-MB-231
cells calculated for evaluating the cytotoxicity of the tested solutions of compound
II.
[0099] After three days of cultivation the control cultures contained (58.7 ± 2.24) × 10
3 cells.
[0100] Cultures, treated with solutions of compound II had the following number of living
cells:
10-11 mol/L: (42.3 ± 2.32) × 103, cell growth inhibition 27.9% (p < 0.001);
10-10 mol/L: (38.1 ± 1.18) × 103, cell growth inhibition 35.1% (p < 0.001);
10-9 mol/L: (34.5 ± 1.94) × 103, cell growth inhibition 41.2% (p < 0.001);
10-8 mol/L: (31.4 ± 1.62) × 103, cell growth inhibition 46.5% (p < 0.001);
4×10-8 mol/L: (31.2 ± 2.33) × 103, cell growth inhibition 46.8% (p < 0.001);
10-7 mol/L: (31.7 ± 1.54) × 103, cell growth inhibition 46.0% (p < 0.001);
10-6 mol/L: (28.4 ± 1.02) × 103, cell growth inhibition 51.6% (p < 0.001).
[0101] It is thus shown, that compound II exerts a significant cytotoxic action against
human breast adenocarcinoma cells. The extent of cell growth inhibition increased
to 51.6% (p < 0.001) when the concentration of compound II was increased.
Example 11. Evaluation of the cytotoxicity of the formulation paclitaxel/compound
II in cultures of human breast adenocarcinoma MDA-MB-231 cell line in relation to
the molar ratio paclitaxel : compound II
[0102] Sodium salt of compound II was converted into the acidic form of compound II and
dissolved in methanol. A solution of paclitaxel in methanol and a solution of compound
II in methanol was mixed. After stirring, the organic solvent was evaporated. The
resulting dried film was dissolved in saline.
[0103] Initial solutions of the formulation in saline at the molar ratios paclitaxel : compound
II equal to 1:3, 1:4, 1:5, 1:6, 1:7 and 1:10 were prepared. From these solutions the
working solutions in MEM with 5% FBS were prepared for adding to cultures. The concentration
of paclitaxel was equal to 10
-6 M.
[0104] Cultures of MDA-MB-231 cells were treated with drug solutions in MEM, containing
5% FBS, after sowing on day 1. Aliquots of the working solutions (2 µL) were added
to 200 µL cultures to a final concentration of paclitaxel in the cultures equal to
10
-8 M. In the control cultures 2 µL of medium with 5% FBS was added as solvent control.
After cultivation for two consecutive days, the number of living cells in the cultures
was calculated and the cytotoxicity of the tested solutions of the formulation was
evaluated.
[0105] After three days of cultivation the control cultures contained (54.4 ± 2.51) × 10
3 cells.
[0106] Cultures, treated with paclitaxel in a concentration of 10 nM, contained (29.3 ±
1.13) × 10
3 cells, thus the cell growth inhibition was 46.1% (p < 0.001).
[0107] Cultures, treated with solutions of the formulation at the molar ratios paclitaxel
: compound II equal to 1:3, 1:4, 1:5, 1:6, 1:7 and 1:10 in medium with 5% FBS, had
the following number of living cells:
1:3: (20.9 ± 1.52) × 103, the cell growth inhibition being 61.6% (p < 0.002), and the cell growth inhibition
compared to that of paclitaxel was increased by 28.7% (p < 0.01);
1:4: (18.6± 1.27) × 103, the cell growth inhibition being 65.8% (p < 0.001), and the cell growth inhibition
compared to that of paclitaxel was increased by 36.5% (p <0.001);
1:5: (17.3 ± 1.16) × 103, the cell growth inhibition being 68.2% (p < 0.001), and the cell growth inhibition
compared to that of paclitaxel was increased by 41.0% (p <0.001);
1:6: (16.8 ± 0.75) × 103, the cell growth inhibition being 69.1 % (p < 0.001), and the cell growth inhibition
compared to that of paclitaxel was increased by 42.7% (p <0.001);
1:7: (16.3 ± 1.20) × 103, the cell growth inhibition being 70.0% (p < 0.001), and the cell growth inhibition
compared to that of paclitaxel was increased by 44.4% (p <0.001);
1:10: (15.9 ± 0.86) × 103, the cell growth inhibition being 70.8% (p < 0.001), and the cell growth inhibition
compared to that of paclitaxel was increased by 45.7% (p <0.001).
Example 13. Evaluation of the cytotoxicity of compound III in cultures of human breast
adenocarcinoma MDA-MB-231 cell line, related to the final concentration of compound
III in the cultures
[0108] The sodium salt of compound III was dissolved in saline (1 mg/ml). From this solution
the working solutions of compound III in MEM with 5% FBS in different concentrations
were prepared by means of consecutive dilutions for adding to cultures.
[0109] Cultures of MDA-MB-231 cells were treated with drug solutions in MEM, containing
5% FBS, after sowing on day 1. Aliquots of working solutions (2 µL) with different
concentrations of compound III were added to 200 µL cultures to final concentrations
of compound III from 10
-11 to 10
-6 mol/L in cultures. In the control cultures 2 µL of medium with 5% FBS were added
as solvent control. After cultivation for two consecutive days, the number of living
cells in cultures was counted and extent of growth inhibition of MDA-MB-231 cells
was calculated for evaluating the cytotoxicity of tested solutions of compound III.
[0110] After three days of cultivation the control cultures contained (54.3 ± 2.12) × 10
3 cells.
[0111] Cultures, treated with solutions of compound III had the following number of living
cells:
10-11 mol/L: (45.1 ± 3.51) × 103, cell growth inhibition 16.9% (p < 0.05);
10-10 mol/L: (45.9 ± 2.84) × 103, cell growth inhibition 15.5% (p < 0.05);
10-9 mol/L: (43.6 ± 2.57) × 103, cell growth inhibition 19.7% (p < 0.01);
10-8 mol/L: (40.3 ± 3.36) × 103, cell growth inhibition 25.8% (p < 0.01);
4 × 10-8 mol/L:(36.5 ± 2.08) × 103, cell growth inhibition 32.8% (p < 0.001);
10-7 mol/L: (35.6 ± 1.68) × 103, cell growth inhibition 34.4% (p < 0.001);
10-6 mol/L: (34.7 ± 1.52) × 103, cell growth inhibition 36.1% (p < 0.001).
[0112] It is thus shown, that compound III alone exerts a significant cytotoxic action against
human breast adenocarcinoma cells. The extent of cell growth inhibition was increased
to 36.1 % (p < 0.001) by increasing the concentration of compound III.
Example 16. Evalution of the cytotoxicity of compound IV in cultures of human breast
adenocarcinoma MDA-MB-231 cell line in relation to the final concentration of compound
IV in cultures
[0113] The sodium salt of compound IV was dissolved in saline (1 mg/ml). From this solution,
the working solutions of compound IV in MEM with 5% FBS in different concentrations
were prepared by means of consecutive dilutions for adding to the cultures.
[0114] Cultures of MDA-MB-231 cells were treated with drug solutions in MEM, containing
5% FBS, after sowing on day 1. Aliquots of working solutions (2 µL) with different
concentrations of compound IV were added to 200 µL cultures to a final concentration
of compound IV ranging from 10
-11 to 10
-6 mol/L in the cultures. In control cultures 2 µL of medium with 5% FBS was added as
solvent control. After cultivation for two consecutive days, the number of living
cells in cultures was counted and the extent of growth inhibition of MDA-MB-231 cells
calculated for evaluation of the cytotoxicity of tested solutions of compound IV.
[0115] After three days of cultivation the control cultures contained (52.7 ± 1.85) × 10
3 cells.
[0116] Cultures, treated with solutions of compound IV had the following number of living
cells:
10-11 mol/L: (44.9 ± 3.02) × 103, cell growth inhibition 14.8% (p < 0.05);
10-10 mol/L: (44.2 ± 3.35) × 103, cell growth inhibition 16.1% (p < 0.05);
10-9 mol/L: (43.6 ± 3.21) × 103, cell growth inhibition 17.3% (p < 0.05);
10-8 mol/L: (39.6 ± 2.74) × 103, cell growth inhibition 24.9% (p < 0.01);
4×10-8 mol/L: (37.1 ± 2.56) × 103, cell growth inhibition 29.6% (p < 0.001);
10-7 mol/L: (36.3 ± 2.08) × 103, cell growth inhibition 31.1% (p < 0.001);
10-6 mol/L: (35.9 ± 2.29) × 103, cell growth inhibition 31.9% (p < 0.001).
[0117] It is thus shown, that compound IV exerts a significant cytotoxic action against
human breast adenocarcinoma cells. By increasing the concentration of compound IV,
it was possible to increase the extent of cell growth inhibition to 31.9% (p < 0.001).
Example 19. Evaluation of the cytotoxicity of compound V in cultures of human breast
adenocarcinoma MDA-MB-231 cell line related to the final concentration of compound
V in cultures
[0118] The sodium salt of compound V was dissolved in saline (1 mg/ml). From this solution
the working solutions of compound V in MEM with 5% FBS in different concentrations
were prepared by means of consecutive dilutions for adding to the cultures.
[0119] Cultures of MDA-MB-231 cells were treated with drug solutions in MEM, containing
5% FBS, after sowing on day 1. Aliquots of working solutions (2 µL) with different
concentrations of compound V were added to 200 µL cultures to a final concentration
of compound V from 10
-11 to 10
-6 mol/L in the cultures. In control cultures 2 µL of medium with 5% FBS were added
as solvent control. After cultivation for two consecutive days, the number of living
cells in cultures was counted and extent of growth inhibition of MDA-MB-231 cells
was calculated for evaluating the cytotoxicity of tested solutions of compound V.
[0120] After three days of cultivation the control cultures contained (54.3 ± 2.12) × 10
3 cells.
[0121] Cultures, treated with solutions of compound V had the following number of living
cells:
10-11 mol/L: (47.1 ± 2.41) × 103, cell growth inhibition 13.3% (p < 0.05);
10-10 mol/L: (46.3 ± 2.49) × 103, cell growth inhibition 14.7% (p < 0.05);
10-9 mol/L: (45.5 ± 2.80) × 103, cell growth inhibition 16.2% (p < 0.05);
10-8 mol/L: (41.1 ± 2.34) × 103, cell growth inhibition 24.3% (p < 0.002);
4×10-8 mol/L (39.6 ± 1.75) × 103, cell growth inhibition 27.1% (p < 0.001);
10-7 mol/L: (39.3 ± 1.22) × 103, cell growth inhibition 27.6% (p < 0.001);
10-6 mol/L: (38.1 ± 1.86) × 103, cell growth inhibition 29.8% (p < 0.001).
[0122] It is thus shown, that compound V exerts a significant cytotoxic action against human
breast adenocarcinoma cells. By increasing the concentration of compound V alone,
it was possible to increase the extent of cell growth inhibition to 29.8% (p < 0.001).
Example 22. Evalution of the cytotoxicity of compound VI in cultures of human breast
adenocarcinoma MDA-MB-231 cell line in relation to the final concentration of compound
VI in the cultures
[0123] The sodium salt of compound VI was dissolved in saline (1 mg/ml). From this solution,
the working solutions of compound VI in MEM with 5% FBS in different concentrations
were prepared by means of consecutive dilutions for adding to the cultures.
[0124] Cultures of MDA-MB-231 cells were treated with drug solutions in MEM, containing
5% FBS, after sowing on day 1. Aliquots of the working solutions (2 µL) with different
concentrations of compound VI were added to 200 µL cultures to a final concentration
of compound VI ranging from 10
-11 to 10
-6 mol/L. In control cultures 2 µL of medium with 5% FBS was added as solvent control.
After cultivation for two consecutive days, the number of living cells in cultures
was counted, the extent of growth inhibition of MDA-MB-231 cells calculated, and the
cytotoxicity of tested solutions of compound VI evaluated.
[0125] After three days of cultivation the control cultures contained (52.7 ± 1.85) × 10
3 cells.
[0126] Cultures, treated with solutions of compound VI had the following number of living
cells:
10-11 mol/L: (46.2 ± 3.54) × 103, cell growth inhibition 12.3% (p > 0.05);
10-10 mol/L: (45.5 ± 2.68) × 103, cell growth inhibition 13.7% (p < 0.05);
10-9 mol/L: (45.3 ± 2.55) × 103, cell growth inhibition 14.0% (p < 0.05);
10-8 mol/L: (40.9 ± 2.88) × 103, cell growth inhibition 22.4% (p < 0.01);
4 × 10-8 mol/L:(39.6 ± 2.70) × 103, cell growth inhibition 24.9% (p < 0.01);
10-7 mol/L: (39.1 ± 2.16) × 103, cell growth inhibition 25.8% (p < 0.001);
10-6 mol/L: (38.3 ± 2.34) × 103, cell growth inhibition 27.3% (p < 0.001).
[0127] It is thus shown, that compound VI exerts a significant cytotoxic action against
human breast adenocarcinoma cells. By increasing the concentration of compound VI,
it was possible to increase the extent of cell growth inhibition to 27.3% (p < 0.001).
[0128] Although the invention has been described with regard to its preferred embodiments,
which constitute the best mode presently known to the inventors, it should be understood
that various changes and modifications as would be obvious to one having the ordinary
skill in this art may be made without departing from the scope of the invention as
set forth in the claims appended hereto.